Cytokinins are phytohormones involved in numerous physiological processes in plants. Plants respond to environmental stresses in part by modifying the relative balance of active and inactive cytokinins. For instance, during times of abiotic stress (which include, but are not limited to, conditions of drought, density, cold, salinity, and/or soil compaction), increased cytokinin oxidase activity shifts the balance in favor of inactive cytokinins, leading to decreased plant productivity. (Jones and Setter, In CSSA Special Publication No. 29, pp. 25-42. American Society of Agronomy, Madison, Wis. (1999)) Conversely, targeted manipulation of the cytokinin balance in favor of active cytokinins could result in increased productivity, even under abiotic stress, through mechanisms such as increased cell division, induction of stomatal opening, inhibited senescence of organs, and/or suppression of apical dominance. (Morris, (1997) In Cellular and Molecular Biology of Plant Seed Development, pp. 117-148. Kluwer Academic Publishers.) In maize subject to unfavorable environmental conditions, cytokinins have been shown to decrease resulting in reduced seed size, increased tip kernel abortion and decreased seed set. (Cheikh and Jones, (1994) Plant Physiol. 106:45-51; Dietrich, et al., (1985) Plant Physiol Biochem 33:327-336). Therefore, these studies show that under stress conditions one approach to improving seed set and seed size would be to maintain the active cytokinin pool above a critical threshold level.
The first naturally occurring cytokinin was purified in 1963 (Letham, (1963) Life Sci. 8:569-573) from immature kernels of Zea mays and identified as 6-(4-hydroxy-3-methylbut-trans-2-enylamino) purine, more commonly known today as zeatin. In the main all naturally occurring cytokinins appear to be purine derivatives with a branched 5-carbon N6 substitutent. (See, McGaw, In: Plant Hormones and their Role in Plant Growth and Development, ed. P. J. Davies, Martinus Nijhoff Publ., Boston, 1987, Chap B3, Pgs. 76-93, the contents of which are incorporated by reference for purposes of background.) While some 25 different naturally occurring cytokinins have been identified, those regarded as particularly active are N6 (Δ2-isopentenyl) adenosine (iP), zeatin (Z), diHZ, benzyladenine (BAP) and their 9-ribosyl (and in the case of Z and diHZ, their O-glucosyl) derivatives. However, such activity is markedly reduced in the 7- and 9-glucosyl and 9-alanyl conjugates. These latter compounds may be reflective of deactivation or control mechanisms.
The metabolism of cytokinins in plants is complex. Multi-step biochemical pathways are known for the biosynthesis and degradation of cytokinins. At least two major routes of cytokinin biosynthesis are recognized. The first involves transfer RNA (tRNA) as an intermediate. The second involves de novo (direct) biosynthesis. In the first case, tRNAs are known to contain a variety of hypermodified bases (among them are certain cytokinins). These modifications are known to occur at the tRNA polymer level as a post-transcriptional modification. The branched 5-carbon N6 substituent is derived from mevalonic acid pyrophosphate, which undergoes decarboxylation, dehydration, and isomerization to yield Δ2-isopentenyl pyrophosphate (iPP). The latter condenses with the relevant adenosine residue in the tRNA. Further modifications are then possible. Ultimately the tRNAs are hydrolyzed to their component bases, thereby forming a pool of available free cytokinins.
Alternately, enzymes have been discovered that catalyze the formation of cytokinins de novo, i.e., without a tRNA intermediate. The ipt gene utilized in the practice of this invention is one such gene. The formation of free cytokinins is presumed to begin with [9R5′P] iP. This compound is rapidly and stereospecifically hydroxylated to give the zeatin derivatives from which any number of further metabolic events may ensue. Such events include but are not limited to (1) conjugation, incorporating ribosides, ribotides, glucosides, and amino acids; (2) hydrolysis; (3) reduction and (4) oxidation. While each enzyme in these pathways is a candidate as an effector of cytokinin levels, enzymes associated with rate-limiting steps have particular utility in the practice of this invention.
One such enzyme is isopentenyl transferase (ipt). An isolated gene encoding ipt was described by van Larebeke, et al., (Nature 252:169-170 (1974); see also, Barry, et al., (1984) Proc. Nat'l. Acad. Sci. (USA) 81:4776-4780 and Strabala, et al., (1989) Mol. Gen. Gen. 216(2-3):388-394). Isolation of ipt genes in Arabidopsis has also been reported. (Takei, et al., (2001) J Biol Chem. 276(28):26405-26410; Kakimoto, (2001) et al., Plant Cell Physiol. 42(7):677-685 and WO 2002/072818; Sun, et al., (2003) Plant Physiol 131:167-176) The invention comprises appropriately modulated expression of ipt genes from any source, including other species, such as maize.
Based on the demonstrable effects of cytokinins in hundreds of experiments across multiple plant species, a transgenic approach to augment active cytokinins in maize could improve its productivity under normal and/or abiotic stress conditions. However, simply increasing the pool of active cytokinins does not automatically lead to enhanced plant growth. In fact, elevating cytokinin levels has been shown to generate detrimental effects on plant phenotype.
For example, Smigocki, et al., (Proc. Nat'l. Acad. Sci. (USA) 85:5131-5135 (1988)), employing the ipt gene from A. tumefaciens operably linked to either the 35S or NOS promoter, showed a generalized effect on shoot organogenesis and zeatin levels. It was noted that the activity of the promoter controls the degree of morphogenic response observed, and unregulated production of cytokinins can result in unwanted pleiotropic effects. With the constructs identified above, undesirable effects included complete inhibition of root formation in tobacco, and stunted cucumber plantlets that did not survive. (Smigocki, et al., (supra); Klee, et al., (1987) Annual Rev. Plant Physiol. 38:467-486).
Attempts followed to express the ipt gene in a more controlled fashion. Medford, et al., (The Plant Cell 1:403-413 (1989)) reported placing the Agrobacterium ipt gene under the control of a heat-inducible promoter and expressing same in transgenic rooted tobacco plants. Levels of cytokinin rose dramatically following heat treatment, and effects observed in transgenics included significant reductions in height, xylem content and leaf size. In both tobacco and Arabidopsis, transgenics displayed slower root growth, disorderly root development and increased axillary bud growth relative to wild-type plants. In addition, the experimental constructs were not satisfactory because the plants exhibited phenotypes associated with excess cytokinin levels, including reduced height, leaf area, and stem width, even in the absence of thermal induction. Further, certain changes were observed in both wild-type and transgenic plants and could be attributed to the heat induction per se.
Schmulling, et al. (FEBS Letters 249(2):401-406 (1989)) transformed tobacco with the Agrobacterium ipt gene under control of the Drosophila hsp70 promoter, which provides a very low level of expression at normal temperatures and a rapid increase in expression after heat shock. Most heat-shocked transgenic calli were greener, had higher cytokinin concentrations, and grew at a more rapid rate than control calli. Plants regenerated from the heat-shocked transgenic calli were described as “fairly normal” and cytokinin levels in these plants did not differ from those measured in wild-type plants. Plants regenerated from uninduced transgenic calli did not differ from controls in either plant phenotype or cytokinin content. A second experiment created callus tissue transgenic for the ipt gene driven by its native promoter. In shoots regenerated from these calli, high cytokinin levels inhibited root formation. These shoots, grafted onto wild-type tobacco stems, displayed tiny leaves and a stunted, highly-branched growth habit. Thus, transformation either resulted in negative phenotypic changes or had no impact.
In PCT Patent Application Publication Number WO91/01323, 7 Feb. 1991, and U.S. Pat. Nos. 5,177,307 and 4,943,674, tomato plants transformed with the ipt gene linked to fruit-specific promoters (2AII, Z130 and Z70) exhibited modified ripening characteristics. Fruits were described as roughened at immature stages, and as mottled, blotchy, and patchy during ripening. See also, U.S. Pat. No. 6,329,570, which discloses transformation of cotton with ipt and a seed-tissue-preferred promoter to modify boll set and fiber quality.
In PCT Patent Application Publication Number WO93/07272, the ipt gene was fused to the chalcone synthase (chs) promoter from Antirrhinum majus and expressed in potato. Phenotypic alterations of transformants included increased tuber yield, plant height and leaf size, thickened stems and delayed leaf senescence. Wang, et al., (Australian J of Plant Phys 24(5):661-672 and 673-683 (1997)) reported increased cytokinin levels in leaf laminae and upper stems of tobacco transformed with ipt driven by a chs promoter, as well as release of axillary buds, inhibition of root development, retardation of leaf senescence, elevation of chlorophyll levels, delay in onset of flowering, retardation of flower development, growth of leafy shoots from the primary root, change in leaf shape, enlarged leaf midribs, enlarged veins, thicker stems, greater node number and increased transpiration rates. Expression of chalcone synthase genes is complex and regulated by a variety of factors, including light, fungal elicitors, wounding and microbial pathogens. In addition, chs expression may be tissue-preferred, occurring in pigmented flowers and roots, and developmentally specific, occurring during early germination. (Ito, et al., (1997) Mol. Gen. Gen. 255:28-37; Shimizu, et al., (1999) Plant Molecular Biology 39(4)785-95).
Additional ipt gene/promoter constructions have been reported.
Smigocki, et al., in WO 94/24848 and U.S. Pat. Nos. 5,496,732 and 5,792,934, disclosed a gene construct capable of conferring enhanced insect resistance comprising a wound-inducible promoter fused to an ipt gene. The study was focused on insect resistance and did not report changes in plant morphology.
Houck, et al., in U.S. Pat. Nos. 4,943,674 and 5,177,307, disclosed several promoters (2AII, Z130 and Z70) coupled with genes encoding enzymes in the cytokinin metabolic pathway, in particular ipt for expression of such enzymes in tomato fruit.
Amasino, et al., in PCT Patent Application Publication Number WO96/29858 disclosed two senescence-specific promoters, including SAG12, operably linked to an ipt gene to inhibit leaf senescence in tobacco. Transformants developed normally, with enhanced biomass and flower and seed production, perhaps owing to the extended developmental period created by the delay in senescence. See also, U.S. Pat. Nos. 5,689,042 and 6,359,197; Gan, et al., (Science 270:1986-1988 (1995)). Jordi, et al., (2000) Plant, Cell and Environment 23(3):279-289, studied the physiological effects of the SAG12:ipt construct in tobacco. While older leaves benefited by retaining chlorophyll, Rubisco, and protein, remobilization of nutrients from older leaves to younger leaves may have been reduced, leading to limited photosynthesis in the upper leaves and restricting potential increases in biomass of these plants, particularly under stress conditions.
Roeckel, et al., (Transgenic Res. 6(2):133-141 (1997)) transformed canola and tobacco with an ipt gene under the control of the developmentally-regulated, seed-specific 2S albumin promoter from Agrobacterium. While ipt mRNA was found only in seeds, and cytokinin levels were evaluated only in seeds, effects of the construct were not limited to seeds: tobacco had reduced roots; canola plants were “surprisingly” (p. 139) taller and had more branches and more seed-bearing structures. However, yield was not affected, nor was leaf type, leaf number, days to first flower, or days to bolting, in either species.
Transformation of tobacco with ipt linked to a copper-inducible, root-specific promoter provided, in 28 of 31 cases, a controlled system for evaluating effects of increased cytokinin production. Morphological changes upon induction included release of apical dominance, increases in total plant leaf number, and delay of leaf senescence. (McKenzie, et al., (1998) Plant Physiol. 116:969-977) Several transgenic lines, however, exhibited uncontrolled cytokinin expression and a radically different, undesirable phenotype, lacking root development and elongation of stems.
Ivic, et al., (Plant Cell Reports 20:770-773 (2001)) reported that expression of ipt in transgenic sugarbeet resulted in severe inhibition of root development, along with undesirable changes in leaf and shoot morphology. Transformed plantlets formed roots slowly or not at all and had a very low survival rate when transferred to soil.
Sa, et al., (Transgenic Research 11(3):269-278 (2002)) reported that transformation of tobacco with ipt from Agrobacterium under the control of a TA29 promoter, which specifically expresses in anthers, resulted in perturbation in the development of anthers and pollen. About 80% of the T0 transgenic plants exhibited a significant decrease in the rate of pollen germination, and up to 20% of the T0 transgenic plants were male-sterile. In addition, abnormal styles and stamens were found in the transgenic plants.
Such negative effects resulting from directed expression of transgenic IPT were noted in PCT Publication Number WO 00/52169: “These approaches also produce undesirable side-effects in the plant and, even in cases where ipt or rolC is expressed under the control of tissue-specific promoters, these side-effects are observed in other tissues, presumably because the cytokinin is transported readily between cells and tissues of the plant.” (Emphasis added).
Thus, there still exists a need for nucleic acid constructs and methods useful in controlling and directing temporally- and spatially-regulated expression of cytokinin metabolic genes in plants, including plant seed and those maternal tissues in which seed development takes place, or in modulating plant sensing of and/or response to cytokinins, in order to improve plant vigor and yield without such detrimental effects as reduced root development or aberrant shoot morphology. This invention provides several such useful nucleic acid constructs and methods to modulate cytokinin activity in plants, including effective levels of cytokinin in plant seeds, developing plant seeds, and related maternal reproductive tissues. Further, the need exists for constructs and methods which can provide said improvements in plant vigor and yield under favorable or unfavorable growing conditions. This invention provides tools and reagents that allow the skilled artisan, by the application of, inter alia, transgenic methodologies, to so influence the level of cytokinin activity, including the metabolic flux in respect to the cytokinin metabolic pathway in seed. This influence may be either anabolic or catabolic, by which is meant the influence may act to increase the biosynthesis of cytokinin and/or decrease the degradation. A combination of both approaches is also contemplated by this invention. Further combinations may include targeted modulation of expression of isolated polynucleotides encoding polypeptides involved in cytokinin recognition and cellular response to provide enhanced cytokinin activity as defined herein.